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. 2017 Jul 21;50(5):e12358. doi: 10.1111/cpr.12358

Figure 5.

Figure 5

Cylindromatosis (CYLD) was targeted by miR‐181d (A) iDCs at day 6 were transfected with miRNA mimics and scrambled control (50 nmol/L), followed by LPS (100 ng/mL) for 24 hours. The targets of miR‐181d were predicted by the bioinformatics tool and were screened by qRT‐PCR. GAPDH was served as the internal control. qRT‐PCR measurements were performed in triplicate (n=3). Data represent the means±SEM (B) Sequence alignment is shown for miR‐181 isomiRs including miR‐181a, b, c and d. Their normalized expression levels iDCs were detected by qRT‐PCR. Data are representative of four independent donors. Data represent the means±SEM. (C) The 3′UTR of CYLD, DUSP10, ESR1 inserts into pmirGLO Dual‐Luciferase miRNA Target Expression Vector. The direct binding between miR‐181d and target genes was detected by Dual‐Glo luciferases reporter assay, and the mimics and luciferase vectors were co‐transfected by transient transfection in 293T cells. Data are representative of three independent experiments. Data represent the means±SEM. (D) Dendritic cells (DCs) were transfected with siCYLD, siESR1 or siDUSP10 (20 nmol/L) for 24 hours on day 6 culture. Expressions of CYLD, ESR1 and DUSP10 were assessed by qRT‐PCR respectively. (E) CD80 and CD83 expressions were detected by qRT‐PCR in LPS‐stimulated DCs, siCYLD, siESR1 or siDUSP10‐treated DCs, compared with N.C.‐treated DCs. (F) Expression of cylindromatosis (CYLD), p‐p65 and p‐65 proteins were detected by Western blotting in DCs transfected with miR‐181d mimics or N.C. respectively. GAPDH was used as the internal control. Morphology of the DCs transfected with N.C. and miR‐181d was examined by Nikon Ti‐U inverted microscope. Data are representative of three independent experiments. Scale bar=20 μm. Data represent the means±SEM. (Student's t tests; *P<.05) (G) Expression of CYLD, p‐p65 and p‐65 proteins were detected by Western blotting in DCs transfected with siCYLD or N.C. respectively. GAPDH was used as the internal control. (H) A schematic representation of miR‐181d regulation of DCs maturation